Effects of four disease-controlling agents (chlorothalonil, CuCl2, harpin, and melatonin) on postharvest jujube fruit quality

Postharvest senescence and disease development can reduce the nutritional value of fresh jujube fruit. Herein, four different disease-controlling agents (chlorothalonil, CuCl2, harpin and melatonin) were separately applied to fresh jujube fruit, and all improved postharvest quality (evaluated by disease severity, antioxidant accumulation and senescence) relative to controls. Disease severity was drastically inhibited by these agents, in the order chlorothalonil > CuCl2 > harpin > melatonin. However, chlorothalonil residues were detected even after storage for 4 weeks. These agents increased the activities of defense enzymes including phenylalanine ammonia-lyase, polyphenol oxidase, glutathione reductase and glutathione S-transferase, as well as accumulation of antioxidant compounds such as ascorbic acid, glutathione, flavonoids and phenolics, in postharvest jujube fruit. The enhanced antioxidant content and antioxidant capacity (evaluated by Fe3+ reducing power) was ordered melatonin > harpin > CuCl2 > chlorothalonil. All four agents significantly delayed senescence (evaluated by weight loss, respiration rate and firmness), with the effect ordered CuCl2 > melatonin > harpin > chlorothalonil. Moreover, treatment with CuCl2 also increased copper accumulation ~ threefold in postharvest jujube fruit. Among the four agents, postharvest treatment with CuCl2 could be considered most appropriate for improving postharvest jujube fruit quality under low temperature conditions without sterilization.

. Disease index in jujube fruit. Effects of chlorothalonil, CuCl 2 , harpin, and melatonin on disease index (%) monitored in jujube fruit after storage for 7, 14, 21, and 28 days at 4 °C. Means associated with the same letter are not significantly different for each day (n = 3; p < 0.05).

Discussion
Infectious disease and senescence can severely decrease postharvest fruit nutrition quality due to antioxidant nutrient loss 26 . In the present work, the nutrition quality of postharvest jujube fruit was evaluated based on disease severity, antioxidant nutrient accumulation, and senescence level. We investigated how the four disease control agents affect postharvest quality of jujube fruit without sterilization. Compared with controls, application   www.nature.com/scientificreports/ of CHT, CuCl 2 , harpin and melatonin significantly attenuated disease severity to varying degrees during storage (Table 1). CHT and melatonin exhibited the strongest and weakest ability, respectively, for disease resistance in plants (Table 1). This shows that organic pesticides (CHT) exhibited greater inhibitory effects on disease development in jujube fruit during storage than did inorganic (CuCl 2 ) and biological (harpin) pesticides, and phytohormones (melatonin). Reports showed that defense enzyme such as PAL and PPO, coupled with GST and GR, play different roles in regulating disease resistance 25 and pesticide degradation in plants 13 . Interestingly, research showed that Cu 27 , harpin 25 and melatonin 28 can strongly stimulate PAL and PPO activities in plants, whereas CHT can profoundly enhance GST and GR activities in plants 12,13 . Thus, the effects of CHT, CuCl 2 , harpin, and melatonin on PAL, PPO, GST, and GR activities in postharvest jujube fruit were investigated (Fig. 1). Compared with controls, application of CHT, CuCl 2 , harpin and melatonin increased PAL, PPO, GST, and GR activities during storage to varying degrees (Fig. 1). However, the highest PAL and PPO activities were observed in melatonin-and harpin-treated jujube fruit during storage (Fig. 1a,b). This also suggests that CHT and CuCl 2 inhibited disease development via a mechanism that is not closely associated with the activities of PAL and PPO, two key enzymes involved in phenolics metabolism 30 . CHT and CuCl 2 may block disease progression by inhibiting glyceraldehyde-3-phosphate dehydrogenase 11 or by generating acute toxic hydroxyl radicals via Fenton reaction 15 , respectively. This suggests that harpin and melatonin may enhance disease resistance by stimulating PAL and PPO activities in postharvest jujube fruit. Interestingly, the highest GST and GR activities were measured in CHT-treated fruits (Fig. 1c,d). These two enzymes play an important role in regulating xenobiotic metabolism but not disease resistance in plants 29 . This suggests that they may contribute to CHT degradation in plants. Moreover, these enzymes are closely associated with antioxidant nutrient (e.g., polyphenol and glutathione) biosynthesis and accumulation  Table 2. Pesticide residues and copper content in jujube fruit. Chlorothalonil residues and copper content (mg kg −1 dry weight) were monitored in jujube fruit after treatment for 0 (water controls for Cu), 7, 14, 21 and 28 days at 4 °C. Means associated with the same letter are not significantly different for each line (n = 3; p < 0.05). www.nature.com/scientificreports/ in plants 30 . Therefore, how these agents affect antioxidant accumulation in postharvest jujube fruit was subsequently investigated. The effects of CHT, CuCl 2 , harpin, and melatonin on antioxidant (e.g., ascorbic acid, glutathione, flavonoids, and phenolics) accumulation and antioxidant capacity (evaluated by Fe 3+ reducing power) were investigated (Fig. 2). Among these antioxidants, phenolics and flavonoids also play a key role in disease resistance 31,32 . Compared with controls, these agents drastically enhanced antioxidant accumulation in jujube fruit during storage (Fig. 2). Interestingly, the antioxidant accumulation level and total antioxidant capacity measured in postharvest jujube fruit following treatment was ordered melatonin > harpin > CuCl 2 > CHT (Fig. 2). Melatonin exhibited the strongest stimulation of antioxidant biosynthesis in postharvest jujube fruit. This may be associated with its antioxidant properties and strong ability to activate antioxidant systems in plants 19 . Similarly, harpin and a trace amount of CuCl 2 also induced antioxidant biosynthesis and accumulation in plants via activation of the antioxidant defense system 23,33 . Compared with controls, application of CHT only slightly enhanced antioxidant accumulation and total antioxidant capacity in postharvest jujube fruit (Fig. 2). Consistently, previous reports have shown that organically grown crops contain higher levels of antioxidant and nutrient compounds than conventionally grown crops exposed to pesticides for disease control 34,35 . Thus, the lowest total antioxidant capacity and highest disease control were monitored simultaneously in CHT-treated jujube fruit during storage (Figs. 1 and 2). This phenomenon could be partly attributed to the toxicity of CHT when binding and depleting cellular glutathione, and it can also inhibit glycolysis by binding to glyceraldehyde 3-phosphate dehydrogenase, leading to cell death 11,36,37 . However, it is also suggested that disease resistance is not closely associated with antioxidant capacity in postharvest jujube fruit. Previous reports showed that free radicals and/or reactive oxygen species (ROS) play a key role in regulating disease resistance 15,24 and antioxidant biosynthesis in plants 38,39 . Therefore, we explored how these agents affect ROS production and accumulation in postharvest jujube fruit.
H 2 O 2 content was determined in postharvest jujube fruit during the first 28 days (Fig. 3). The results showed that CHT, CuCl 2 and harpin rapidly induced H 2 O 2 accumulation in jujube fruit after treatment for 1 day (Fig. 3). Moreover, the results show that CHT, CuCl 2 and harpin play a role, as oxidant-like inducers 32 , in stimulating antioxidant biosynthesis and accumulation via ROS production in jujube fruit during storage (Fig. 3). However, more evidence is required to determine the detailed mechanism. By contrast, melatonin inhibited H 2 O 2 overproduction, and no peak was observed during the whole storage stage (Fig. 3). However, the highest antioxidant accumulation was measured in melatonin-treated jujube fruit (Fig. 2). This indicates that melatonin, which acts as a phytohormone, can efficiently regulate antioxidant biosynthesis and accumulation via a different mechanism [19][20][21] .
Antioxidants (e.g., ascorbic acid and glutathione) also play a key role in delaying fruit and vegetable senescence during storage 39 . Therefore, we next investigated how these agents affect postharvest jujube fruit senescence (Fig. 4). Compared with controls, treatment with these agents delayed jujube fruit senescence (evaluated by weight loss, respiration rate and firmness) to varying degrees (Fig. 3). The senescence-delaying effects of these agents were ordered CuCl 2 > melatonin > harpin > CHT (Fig. 4). This order is similar to that of antioxidant accumulation and antioxidant capacity: melatonin > harpin > CuCl 2 > CHT (Fig. 2). This shows that the senescencedelaying effects are closely associated with antioxidant accumulation in postharvest jujube fruit (Figs. 2 and 4). This is consistent with published data 6,7,9,23 , which showed that antioxidant capacity made a key contribution to delaying senescence of postharvest fruit and vegetables. However, CuCl 2 rather than melatonin exhibited the greatest senescence-delaying effects (Figs. 2 and 4; p < 0.05). This phenomenon could be partly attributed to the inhibitory effects of copper-induced oxidative stress on aquaporin, a key channel for water permeability in plants 40 . Consistently, the lowest decline in weight loss was observed in CuCl 2 -treated jujube fruit during storage (Fig. 4b). Compared with CHT, melatonin and harpin drastically delayed postharvest fruit senescence to varying degrees (Fig. 4). One plausible explanation is their ability to induce antioxidant accumulation in plants 19,23 . However, CHT exhibited the lowest ability to delay postharvest fruit senescence among the four agents (Fig. 4). This also showed that antioxidant capacity but not disease control ability makes a greater contribution to delaying postharvest senescence in jujube fruit.
Among the four disease control agents, the pesticide CHT exhibited the strongest inhibitory effects on disease development, but this was coupled with the lowest antioxidant content and ability to delay senescence in postharvest jujube fruit. Moreover, Table 2 shows that pesticide (CHT) residues were detected in jujube fruit even after storage for 4 weeks 14 . This poses a great threat to human health and the environment. By contrast, melatonin and harpin achieved higher antioxidant levels but exerted lower inhibitory effects on disease development. Interestingly, CuCl 2 application not only drastically reduced disease severity but also induced antioxidant biosynthesis and accumulation 41 . In addition, CuCl 2 treatment enhanced copper accumulation (~ threefold) in jujube fruit, relative to controls ( Table 2). It is known that trace amounts of copper are required for human health 42 . Thus, application of CuCl 2 may perform better than applying other agents such as CHT, harpin, and melatonin in terms of simultaneously controlling disease, delaying senescence, and increasing antioxidant accumulation in postharvest jujube fruit without sterilization.
In conclusion, the tested agents enhanced disease control and bioactive compound accumulation, and delayed senescence in postharvest jujube fruit to varying degrees. Among the disease control agents tested, CHT performed best for disease control, but worst for delaying senescence and promoting antioxidant accumulation. By contrast, melatonin performed best for enhancing antioxidant capacity, but worst for disease control. However, delaying jujube fruit senescence and inhibition of disease development could be efficiently achieved by CuCl 2 treatment. This showed that antioxidant capacity was closely associated with postharvest senescence, but not closely associated with disease control ability. Copper, the key component of the traditional pesticide Bordeaux mixture 15 , was tested for its ability to efficiently control disease development, increase antioxidant accumulation, and delay senescence in postharvest jujube fruit. The results provide a simple method for improving postharvest jujube fruit quality under low temperature (4 °C) without sterilization. This phenomenon could be partly attributed to the different roles of these agents in regulating the content of H 2 O 2 , which is closely associated with

Materials and methods
All local, national or international guidelines and legislation were followed during the course of this study.  43 . Fruits without visible defects were chosen based on uniform shape and appearance. Postharvest jujube fruits without sterilization were divided into five groups (water control, CHT, harpin, CuCl 2 , and melatonin) and used in subsequent experiments. Treatments were performed under low temperature conditions (4 °C, 70% humidity) as follows:

Reagent preparation. Harpin protein and chlorothalonil (Daconil
Step 1: fruit were immersed in distilled water (controls), chlorothalonil (10 mM), harpin (30 mg L −1 ), CuCl 2 (0.5 g L −1 ) or melatonin (0.1 mM) for 2 h; Step 2: treated fruit were washed with distilled water and dried in air at 25 °C for 2 h; Step 3: treated fruit were stored at 4 °C and 70% relative humidity for up to 28 days. One hundred samples were used each week to evaluate weight loss, firmness, respiration rate and disease index. Additionally, other samples of jujube fruits were stored for 7, 14, 21 and 28 days at 4 °C prior to analysis of CHT and copper accumulation, ascorbic acid content, glutathione level, total phenolics and flavonoids content, total antioxidant capacity, and measuring enzyme activities for PAL, PPO, GR, and GST activities.
Herein, only edible fruit tissues (including flesh and peel) were collected for parameter analysis. For each assay, three replicates were performed for each treatment, each including 20-30 fruits.
Copper assay. Jujube fruits were collected, washed, air-dried, and ground into powder with a mortar and pestle. For heavy metal extraction, digestion tubes were thoroughly acid washed and dried 8 . Dry powdered sample (1 g) was placed in a digestion tube and 10 mL of HNO 3 , HClO 4 and H 2 SO 4 (5:1:1) were added and incubated for 12 h. The tubes were then placed in a digestion block at 80 °C for 1 h, and the temperature was slowly raised to 120-130 °C. When digestion was completed, the solutions were cooled, filtered, and diluted to 100 mL with double-deionized water. Cu in filtrates was assayed using an Analyst 700 atomic absorption spectrometer (Perkin Elmer, USA).
Pesticide residue assay. Pure chlorothalonil was obtained from Macklin Biochemistry & Technique Company (Shanghai, China). Pesticide residues were extracted and determined in 5 g of chopped fruit tissues 7 . Jujube fruits combined with petroleum ether (PE) and anhydrous sodium sulphate (ASS) were homogenized in a highspeed disperser (12,000 × g for 5 min). A Büchner funnel (7 cm) containing 10 g of ASS was used for filtration of the fruit mixture, and 50 mL of redistilled PE was used to wash the filter cake three times. The filtrates were mixed in a flat-bottomed flask (0.5 L) and dried with an N 2 stream. Chlorothalonil was dissolved in redistilled PE, and 5 mL of solution was subjected to quantitative analysis by a gas chromatography instrument (GC-14C, Shimadzu, Japan) equipped with a phosphorus filter and a flame photometric detector.
Disease index assay. The disease index was used to evaluate the development of disease resulting from natural infection. It was assessed by monitoring the growth of visible pathogen lesions on the jujube fruit surface as described previously 44 . Disease index is divided into five levels: grade 0, no lesions; grade 1, some lesions; grade 2, lesion area < 25%; grade 3, lesion area 25-50%; grade 4, lesion area > 50%. Disease index was assessed by measuring the lesion area on each fruit pericarp and calculated using the equation: Weight loss, firmness, and respiration rate analyses. Weight loss was evaluated by weighing each jujube fruit before and after the storage period, and presented as the percentage weight loss compared to initial weight. Firmness was measured using a GY-3 pressure tester (Aidebao Instrument Co. Ltd, Leqing, China) equipped with an 8 mm diameter probe. Decay incidence is the number of fruits showing decay symptoms relative to the total number of fruits in each treatment (expressed in %). Respiration rate was estimated using a previously described method 45 with some modifications. In each treatment, 10 jujube fruits (water, chlorothalonil, harpin, CuCl 2 or melatonin) were randomly sampled and sealed in a glass container with 0.02 L 0.4 M NaOH at room temperature for 1 h. Next, 0.01 L saturated BaCl 2 and three drops of phenolphthalein were added, and the solution was titrated with 0.1 M oxalic acid until the red color disappeared. The respiration rate of samples is expressed as mg kg − www.nature.com/scientificreports/ Antioxidant measurement. The titrimetric method with 2,6-dichloro-phenol-indophenol (2,6-DPI) was used to assess the ascorbic acid content 7 . Briefly, 1 g of homogenized fresh jujube fruit was mixed with 20 mL of 2% oxalic acid. The mixture was homogenized, diluted to 0.1 L with 2% oxalic acid, and filtered. Next, 10 mL of filtered solution was titrated with 0.01% 2,6-DPI solution. The endpoint was considered reached when the solution had been pink in color for 15 s. Calibration of the 2,6-DPI solution was performed using a 0.05% ascorbic acid solution. Results are expressed as mg ascorbic acid equivalents per g of fresh weight (mg g −1 ). Glutathione was determined by an enzymatic cycling assay described previously 46 . Oxidized glutathione (GSSG) was measured after removal reduced glutathione (GSH) by 2-vinylpyridine derivatization. GSH was determined by subtraction of GSSG from total glutathione (GSH + GSSG).
Total phenolics were measured using the Folin-Ciocalteu reagent method 47 ; the absorbance was recorded at 760 nm using a UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan) and results expressed as gallic acid equivalents (mg g −1 of dry weight).
The total flavonoid content was determined by the aluminum chloride colorimetric method 48 using catechin as a standard, and expressed as mg of catechin equivalent (CE) per kg of dry weight.
Total antioxidant capacity assays were performed described previously 49 . Fruits were ground to powder in liquid N 2 using a mortar and pestle, 5 g of fruit tissue powder was transferred to 1 L of 80% (w/v) methanol-water solution, and incubated at room temperature for 2 h in the dark. Extracts were filtered, filtrates from each replicate were pooled, and solvent was removed under vacuum at 45 °C using a rotary evaporator. Crude extracts were then stored in a desiccator at 4 °C for subsequent total antioxidant capacity analysis using the ferric reducing ability of plasma (FRAP) assay. FRAP reagent comprised 10:1:1 (v/v) 100 mM acetate buffer (pH 3.6), 20 mM FeCl 3 solution and 10 mM 2,4,6-tripyridyl triazine solution in 40 mM HCl. FRAP reagent was prepared and warmed to 37 °C in a water bath just before use. Samples (50 µL) were added to 1.5 mL of FRAP reagent and the absorbance of the reaction mixture was recorded at 593 nm after 5 min using a UV-Vis spectroscopy instrument (Shimadzu). A standard curve was constructed using FeSO 4 solution and results expressed as mM Fe(II) g −1 dry weight of jujube fruit.
Hydrogen peroxide assay. Hydrogen peroxide (H 2 O 2 ) accumulation in jujube fruits was measured using